7900ht cycler

RNA isolated with an RNeasy kit (Qiagen) was reverse-transcribed into cDNA with Superscript III (Invitrogen). Power Syber Green Master Mix (Applied Biosystems) was used with primers described in Supplementary Table S2. Quantitative real-time PCR was performed on an Applied Biosystems 7900 HT cycler (Supplementary Methods). The product of expression of three M1 markers (Nos2=inducible nitric oxide synthase, CXCL10, and IL-1β) was divided by the product of expression of three M2 markers (Arginase 1, TGF-β, and MMP9) for M1/M2 ratios and inverted for M2/M1 ratios.

RNA isolation and quantitative RT-PCR (RT-qPCR) were essentially performed as recently described (22 (link),44 (link)). In brief, total RNA extracted using Trizol served as template for cDNA synthesis by random priming. RT-qPCR was performed based on SYBRgreen I technology using SYBR Select Master Mix (Life Technologies) in a 7900HT-cycler (Applied Biosystems). Whenever possible, primer pairs spanning an exon/exon border were selected using the Primer Blast database (http://www.ncbi.nlm.nih.gov/tools/primer-blast/). For non-exon-border spanning primer pair, no-RT controls were performed. For all primer pairs an annealing temperature of 58°C in a three step protocol was used. Relative changes of RNA abundance were determined by the ΔΔCt method using ACTB and GAPDH for normalization, as previously described (44 (link)). For primers used see Supplementary Table S5.

To isolate total RNA from cells, we used Trizol (Invitrogen). Preparations of <2 μg of total RNA were treated with DNase I (Thermo Fisher Scientific), according manufacturer instructions. For first-strand cDNA synthesis, we used random octamers and oligo dT-16 primers and MuLV reverse transcriptase (High capacity RNA-to-cDNA kit, Thermo Fisher Scientific). PCR amplification was limited to 30 cycles with 60°C annealing temperature. We separated the PCR products on 3% agarose gel. PCR primer sequences are provided in the Supplementary Table S1. All experiments were repeated three times.
Quantitative reverse transcriptase-PCR (qRT-PCR) was performed using SYBR Green master mix (Applied Biosystems) in a 7900 HT cycler (Applied Biosystems). qRT-PCR primers are listed in Supplemental Table SII. The quantification of the gene expression was performed using the ΔΔC_T method. Relative levels of splice isoforms are presented as a ratio of mRNAs, with exon 242 included, versus mRNAs, with exon 242 skipped. The fold change in inclusion/skipping ratio was obtained when compared to the control (transfection with RBM20 expressing plasmid). n = 3 for all samples, all data are expressed as mean ± SEM. Group comparisons were analyzed by one-way ANOVA and Bonferoni post test. P values were considered statistically significant as follows: *P < 0.05; **P < 0.01; ***P < 0.001.